Discoverybio wide pore c18 column

The extraction of wheat proteins was done according to the stepwise quantitative procedure of Wieser et al. [44 (link)]. Whole meal flour (100 mg) was extracted stepwise with 0.4 M NaCl (albumins and globulins, AG), 50% 1-PrOH (GLI), and 50% 1-PrOH + 2 M urea + 0.05 M of Tris-HCl (pH = 7.5) + 1% DTT (GLU). Protein separation was carried out using the Perkin Elmer LC 200 chromatograph controlled by the TotalChrom software (Perkin Elmer Instruments, Waltham, USA) on a Discovery BIO Wide Pore C18 column (300 Å pore size, 5 μm particle size, 4.6 × 150 mm i.d.) (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). The mobile phase consisted of millipore water with 1% trifluoroacetic acid (v/v) (A) and acetonitrile with 1% trifluoroacetic acid (v/v) (B). The column temperature was 50 °C and injection volume was 20 µL. The gradient elution profile was as follows: from 24%–54% B in 30 min, isocratic at 90% B for 5 min, returning to the initial conditions in 5 min, and column equilibration in 5 min. The flow rate was controlled at 1.0 mL/min. The peaks were detected at 210 nm with a photodiode array detector. The peak areas under AG, GLI, and GLU chromatograms were summed and used as a direct measure of total content of extractable wheat proteins and consequently, the proportions (%) of protein fractions and single protein types were calculated [44 (link)].

The wheat protein extraction from 100 mg of flour sample was done stepwise accordingly to the procedure of Wieser et al. (1998) [31 (link)]. Proteins separation was carried out using Perkin Elmer LC 200 chromatograph controlled by Total-Chrom software (Perkin Elmer Instruments, Waltham, MA, USA) on a Discovery Bio Wide Pore C18 column (300 Å pore size, 5 μm particle size, 4.6 × 150 mm i.d.) (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). Amounts of 0.1% trifluoroacetic acid (TFA) in water (v/v) and 0.1% TFA in acetonitrile (ACN) were applied as mobile phase and 20 μL samples were injected for analyses. AG, GLI, and GLU fractions were eluted with a linear gradient from 24 to 58% ACN over 30 min at a 1 mL min⁻¹ flow using a column temperature of 50 °C. All determinations were made in duplicate. The peak areas under AG, GLI, and GLU chromatograms were summed and used as a direct measure of total content of extractable wheat proteins. Consequently, the proportions (%) of protein fractions and single protein types were calculated [38 (link)].